Working with conscious animals is a requirement of important biomedical research techniques such as infusion, in vivo microdialysis, in vivo ultrafiltration, in vivo electrochemistry, biosensors and electrophysiology. All of these methods study the functioning of living organs such as the brain, heart, circulatory system, muscles, etc. They also involve connections between external devices such as syringe pumps, fraction collectors, electrometers, vacuum sources, light sources, and potentiostats to implants in the animal's body such as infusion cannula, ultrafiltration probes, microdialysis probes, or electrodes. In conjunction with these tests, it is sometimes desirable to monitor animal activity and/or feces and urine composition. The means of connection is typically a length of flexible, hollow, plastic tubing, a flexible wire, or an optical fiber.
Frequently, in the prior art, the connection of one or more lines of tubing for conveyance of fluids in such tests involves the use of a liquid swivel, or, for electric or optical leads, the use of a swivel-commutator (such as an electrocannular device). In general, a portion of the lead is connected to the top of the swivel which is mounted on a support above the animal, while an additional portion of the lead is connected from the implant on the animal to the underside of the swivel. Liquid swivels are designed so that the top and bottom half rotate independently and an internal seal connects the two halves. When the connection is electrical or optical, a form of commutator is required. For liquid swivels and swivel commutators, the lead is discontinuous, i.e., it is somehow "split" at the swivel, so that the bottom half of the lead may be required to rotate with respect to the top half of the lead.
Liquid swivels are frequently unreliable. Further difficulty in their employment results when there is a need to connect more than one tubing line, as in microdialysis. Multi-channel liquid swivels typically use concentric cannulae with concentric, complex seals separating each channel from the next. The seals wear easily when exposed to salty, physiological solutions. When they leak, cross-channel contamination is a common occurrence. Use of liquid swivels is also difficult when an electric or optical line (lead) is to be connected to the animal, for such an electric or optical lead requires the addition of a commutator to maintain contact with leads attached to the animal. Although the swivel and commutator can accommodate rotation of the respective leads, the leads, or a portion thereof, can become entangled when the leads rotate with respect to each other.
The use of a liquid swivel undesirably adds additional volume to a fluid path. For example, connecting tubing for microdialysis typically has an internal diameter of 0.12 mm. A length of 10 cm of such tubing contains a volume of approx. 1.2 .mu.L of fluid. A two channel liquid swivel, such as the stainless steel model available from Instech Laboratories of Plymouth Meeting, Pa., has a dead volume of 1.4 .mu.L for the center channel and 18.5 .mu.L for the side channel. Thus, when such a swivel is used, it takes more time to transfer fluid the same distance due to the dead volumes of the channels. Consider a situation where the distance between the animal and a device such as a fraction collector is 30 cm, where the fluid travels at a rate of 2 .mu.L per minute, and the height of the swivel is 5 cm. The volume in 30 cm of tubing is 3.6 .mu.L, and it would take 1.8 minutes for the fluid to travel from one end to the other of this 30 cm tubing. A 5 cm long swivel and 25 cm of tubing would occupy the same distance, but the volume of this combination (using the center swivel channel of the Instech Laboratories tubing ) would increase to 4.4 .mu.L, and, thus, it would take 2.2 minutes for fluid to traverse this combination of tubing and swivel. If the side channel of the swivel was used, the volume would be 21.5 .mu.L, and time for traversing the tubing and swivel would increase to 10.7 minutes.
The use of liquid swivels adds further limitations when the fluid within the system is blood. Use of liquid swivels generally requires the use of heparinized saline as a wash fluid for blood sampling since the swivel in contact with the blood leads to clotting in the absence of heparin. Consequently, blood serum testing is unavailable in a system utilizing a liquid swivel due to the presence of heparin. Additionally, the injection of heparinized saline into the animal affects the PK behavior of drugs and can lead to increased bleeding in the animal. If a method of blood sampling is used in which the animal is not injected with heparinized saline, there is a risk of dehydration of the animal, consequently, the blood sampling activity is significantly restricted.
The use of liquid swivels is common, as described above. The article "Triple electrical channels on a triple fluid swivel and its use to monitor intracranial temperature with a thermocouple" by Parada et al., Journal of Neuroscience Methods, Vol. 60 (1995), pg. 133-139, describes a complex liquid swivel which deals with the aforementioned limitations in fluid channels by creating an extremely complex device. It is desirable to avoid such a complex system, and to avoid the use of liquid swivels while permitting for free movement of the animal.
Another shortcoming of swivel systems relates to tracking the movement of the freely-moving animal. Rotational and vertical behavior in laboratory rodents are well-established indicators of neurochemical changes occurring in the animal during testing. The clockwise or counterclockwise preference of the animal, the frequency of such rotation, and similar information concerning the vertical movement of the animal are valuable data not available with the prior art liquid swivel systems. It is therefore desirable to provide a system which not only permits for rotation of the animal, but also which is capable of tracking the rotational and vertical movements of the animal for identification of behavioral changes occurring during testing.
The use of liquid swivels and commutators also results in additional manufacturing costs and in unwanted repair costs. Because the swivels naturally wear out during the course of normal use, continual repair or replacement of the swivel is required. It is preferred to avoid the use of swivels to avoid the extra expense thereof in manufacture and repair, but to maintain the ability to perform operant and metabolic testing of an animal with test leads connected.
An apparatus for infusion in a freely-moving animal is disclosed in U.S. Pat. No. 3,897,751, Gullino et al. The apparatus of Gullino et al. utilizes a continuous catheter to infuse the animal and permits for movement of the animal by threading the catheter between the walls of the cage and an elevated cover. Gullino et al. does permit for rotation of the animal by use of a spring. However, if such rotation were to occur, because of the inherent tension of a spring, stress is applied to the catheter. Such rotational stress could result in harm to the animal or may disconnect or impair the connection of the catheter to the source of fluid. Therefore, it is desired to provide an apparatus for infusion in a freely-moving animal which permits for the rotational movement of the animal while employing continuous leads which does not result in harm to the animal or disconnection or impairment of the connection of the lead to its source.
Another apparatus for conducting tests on freely-moving animals is described in a scientific article entitled "A novel apparatus that permits multiple routes for infusions and body-fluid collections in a freely-moving animal", Matsumura et al., Journal of Neuroscience Methods, Vol. 57 (1995), pg. 145-149. Matsumura et al. does disclose a movement-responsive apparatus which permits for rotation of the animal by rotating the floor of the cage housing the animal. Specifically, in the disclosed apparatus, multiple fluid lines are passed through the center of a device mounted to a fixed support above a cylindrical chamber. The animal is tethered to this device by the electrical lines. The electrical lines are connected through a slip-ring commutator on the exterior of the device. This type of connection means that the top and bottom half of the device rotated independently, like a swivel. The floor of the cylinder portion of the cage is moved in response to the animal's movement while the walls of the cage are immobile.
The apparatus of Matsumura et al. has several shortcomings, however. For example, the invention of Matsumura et al. permits full rotation of the animal through three, or more, complete 360.degree. turns before responding to the animal's movement with counterrotation. This movement can create undesirable twisting and stress on the leads connected to the animal. In addition to the potential for equipment malfunctions, the twisting and stress can cause discomfort to the animal thereby altering the animal's behavior. Further, despite employment of a microcomputer, the apparatus of Matsumura et al. does not track rotational or vertical behavior of the animal--valuable indicators of neurochemical changes occurring in the animal during testing.
A method and apparatus for conducting automated microsampling of blood in conscious rodents is described in a scientific book entitled Neuroscience Research Methods, Clark et al., Vol. 1, Chapter 10, pages 205-222. Clark et al. teaches a method and apparatus for automated microsampling of blood which uses a Gilson Minipuls 3 peristaltic pump to withdraw blood from the animal through a sterile polyethylene catheter. The blood is then transported through nonsterile polyethylene tubing which is connected to the catheter using silastic tubing. Within the length of tubing is positioned a liquid swivel to permit the animal to rotate without twisting the tube. Lee valves are used in conjunction with the peristaltic pump to direct the blood sample to chilled open vials. In this example, the Lee valve is representative of a valve system in which the blood is in direct contact with valve components, in which the blood departs from the conducting tube when entering the valve, and in which blood returns to another conducting tube when exiting the valve. The use of polyethylene tubing, a swivel and Lee valves mandates the use of heparinized saline as a wash fluid to reduce the risk of clotting within the tube, the swivel, and the valves.
The method and apparatus of Clark et al. has several shortcomings. For example, the shortcomings associated with use of a swivel as discussed supra apply to Clark et al. Also, use of a peristaltic pump for control, is a high cost solution, partially due to the added costs for calibration of individual systems. The ability to precisely recreate certain experiments is limited due to volumetric errors associated with peristaltic pumps which deliver a pulsatile flow and are subject to degradation of the peristaltic tubes during use--a change which also affects flow rate. Additionally, sterile and non-sterile polyethylene tubes and catheters are subject to clotting. Once a tube or catheter is clogged, it must be removed, a replacement piece of tube or catheter must be cut, measured, and installed, and the system must be recalibrated. Further, silastic tubing connections are known to become loose resulting in leakage and/or clotting. More problems result from having valves in contact with blood. First, the potential for clotting exists. Second, once the valve is in contact with blood it cannot be conveniently re-sterilized. Consequently, the valve must be discarded after a single application. Finally, the use of heparinized saline as a wash fluid creates the risk of introducing heparin into the animal which can result in bleeding and can affect the PK behavior of drugs. Additionally, the presence of heparin eliminates the possibility of collecting blood serum since clotting is required in order to collect the serum by centrifuging.
Systems for monitoring the vertical activity of animals exist in the prior art. A typical system is embodied in the Harvard/Columbus Instruments Basic Activity Meters (Harvard apparatus, Natick, Mass.). These units use infrared sensor beams at spaced intervals in conjunction with a control unit to detect activity. These systems are bulky and expensive. More exotic units such as the Harvard/Columbus Instruments Activity Monitoring System (Harvard apparatus, Natick, Mass.) utilize an infrared scanner to monitor animal movements at even greater costs. It is desired to provide an apparatus for monitoring vertical movement in a freely moving animal which is inexpensive, compact and significantly less complex.
Accordingly, the avoidance of the use of a liquid swivel in a conscious animal monitoring system thereby results in the following advantages over the prior art:
Eliminates the severe limitation in the number of viable fluid channels connected to a test animal due to increasing friction loads as more channels are added to a liquid swivel which eventually render the swivel immobile; PA1 Eliminates the need to compensate for and accommodate the extra system volume of a liquid swivel; PA1 Imposes no restrictions on the number or combination of electrical, fluid or optical channels employed in the system; PA1 Imposes no restrictions on the relative placement of different types of channels (e.g. electrical and fluid) used in the same monitoring system; PA1 Eliminates the need to compensate for and accommodate the extra liquid travel time between the implant and the external device (pump, fraction collector, etc.) caused by a liquid swivel; PA1 Avoids cross-contamination between channels that occurs in a liquid swivel which is capable of handling multiple fluids; PA1 Avoids the extra expense from continual replacement or repair of swivels which naturally wear out during the course of normal use. PA1 Connecting tubing, optical fibers, and wires (collectively referred to herein as "leads") to are not broken. They remain as a single, unbroken piece connecting the implanted device to the external device. PA1 When tubing must be broken to allow for multiple paths such as is the case in automated micro blood sampling, tubing and connectors which are easily coated with an anti-coagulant can be used, eliminating the need for heparinized sterile saline solution. PA1 Multiple leads can be used with no risk of cross-contamination since they are not joined or connected through a swivel, commutator or other junction. PA1 Electrical wires and/or optical fibers can be used at the same time as tubing lines filled with fluid. PA1 The rotational sensor assembly differentiates between clockwise and counterclockwise rotation by the animal and moves the animal, in its cage, in the opposite direction. PA1 The vertical sensor assembly provides an inexpensive and reliable means for detecting vertical movement of the animal. PA1 Signals from the rotational sensing means and the vertical sensing means can be recorded by a simple strip chart recorder or device such as a computer. These signals record the overall activity of the animal, its clockwise and/or counterclockwise movement and vertical movement. PA1 The rotational sensor assembly is mounted on a counter-balanced arm so that slack in tubing or wires is taken up and away from the animal. This also creates less stress for the animal since the system responds to its vertical movements. Further, the system is not likely to cause behavioral changes in the animal. PA1 The container can serve as an operant behavior and/or a metabolic container. PA1 The AMBS apparatus can be used without heparinized saline allowing blood serum testing while eliminating effects on PK behavior of drugs or potential for bleeding. PA1 The automated micro body fluid sampling apparatus uses syringe pumps which provide more precise control over sampling evolutions, thereby reducing errors and giving a higher degree of repeatability than prior art systems. PA1 The automated micro body fluid sampling apparatus uses a personal computer or other controlling device to automate the sampling process thereby realizing simplicity in setup, expanded control options, expanded data collection possibilities, and expanded data presentation alternatives. These expanded capabilities include the potential for real time display of experiment results on the internet. PA1 The automated micro body fluid sampling apparatus makes it possible to do several different types of experiments in a concurrent fashion, instead of in a serial or stepwise fashion, thus saving the researcher time and laboratory space.
It is another advantage of the present invention to provide a movement-responsive system which tracks rotational and vertical behavior of the animal, while imposing no restrictions on operant or metabolic testing.
It is yet another advantage of the present invention to provide a system which does not require leads subject to rotational forces to be affixed to either the animal or the source or device to which the lead is connected to thereby avoid potential harm to the animal or of disconnection or impairment of the connection of the lead to the animal or to the source or device.
It is still another advantage of the present invention to provide sensor systems which are inexpensive to manufacture and which are highly reliable during operation.
Further, it is still another advantage of the present invention to provide a movement-responsive system which can be utilized for a myriad of biomedical tests performed on freely-moving animals, including but not limited to infusion, electrophysiology, blood monitoring, ultrafiltration, microdialysis, electrochemistry, optical fiber transmission, operant behavior, pharmacokinetics, bile sampling, automated micro blood sampling, and metabolic and behavioral monitoring and which permits for more than one such test to be performed concurrently.
Another advantage of the present invention is to provide a testing apparatus which does not result in undesirable change in the animal's behavior as results from some of the prior art systems.
An additional advantage of the present invention is to accommodate both the rotational and vertical movements of an experimental animal by use of a rotational sensor mounted on a counter-balanced arm and tether assembly which keeps leads out of the animal's reach, and reduces animal stress by minimizing collar tension.
It is also an advantage of the present invention to provide a method and apparatus for automated micro blood sampling which eliminates the use of heparinized saline solution while reducing the risk of clotting and leaking. This same apparatus can be used to sample other body fluids such as bile which can be removed from the bile duct and replaced with an equivalent volume of bile salts solution.
It is another advantage of the present invention to provide a system with these and other capabilities and features which is inexpensive to manufacture, repair and maintain.